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Annular Beam Shaping in Multiphoton Microscopy to Reduce Out-of-Focus Background 时间:2017-08-04 13:57 来源:未知作者:admin 点击: 次 Abstract:Despite the inherent spatial confinement of multiphoton processes that arises from focusing through an objective, the maximum imaging depth in conventional multiphoton microscopy is ultimately limited by noise from out-of-focus fluorescence. This is particularly evident when imaging beyond shallow depths in highly scattering tissue as increased laser powers are necessary. The out-of-focus signal originates from multiphoton processes taking place primarily at shallow depths and deteriorates contrast and limits imaging depth. In this paper, annular laser beams are explored as a concept to reduce this background signal in multiphoton microscopy. The approach is theoretically verified by data from simulations and proof of principle is demonstrated on a custom-built experimental multiphoton microscopy platform. Annular laser beams were created by adopting wavefront control using a spatial light modulator and implemented for imaging tissue phantoms simulating turbid media and human skin ex vivo. The signal-to-background ratios were calculated and compared to images acquired with a traditional, filled-aperture Gaussian beam. Experiments in tissue phantom show an improvement in signal-to-background ratio of about 30% when using annular beam illumination in comparison to Gaussian illumination at specific depths. When laser power is not the limiting factor, this approach is expected to provide even greater benefits.
1. Introduction
Multiphoton microscopy (MPM) has evolved from a photonic novelty [1] to a well-established laboratory tool that allows for noninvasive 3D imaging of tissue [2]. Since MPM is operating in the “optical window” of biological tissue (~600–1300 nm), it allows for increased imaging depths compared to single photon excitation modalities like confocal microscopy [3]. MPM utilizes high intensity fs-pulse lasers to generate a high flux of photons necessary for multiphoton absorption. Conventionally, these nonlinear processes are assumed to occur only at the focal volume where extremely high photon flux is generated, allowing for noninvasive three-dimensional optical sectioning. Recently, MPM has enabled powerful applications in life sciences such as high-speed cell mitosis imaging [4], real-time lymphocyte tracking [5], and cell migration monitoring [6]. MPM has also been established as a tool for visualization in turbid biological matter such as the human skin [7, 8]. For dermatological purposes, multiphoton microscopy has been commercialized for clinical use (DermaInspect, Jenlab, Germany) [9]. In addition, in vivo MPM is advancing the field of neuroscience by allowing for optical visualization of neural architecture and functions [10, 11].
Assuming sufficient excitation power and aberration control, the fundamental factor limiting imaging depth when performing MPM in tissue is the background fluorescence originating from out-of-focus areas above the imaging plane [12–14]. The out-of-focus signal is a result of increasingly high excitation powers that are necessary to maintain high excitation intensity when the focal volume is located deep in the tissue. This background signal deteriorates the imaging contrast (signal-to-background ratio) and in turn limits imaging depth. Thus, efforts should be made to minimize this undesired signal, to improve the signal-to-background ratio and thereby increase the imaging depth. The implementation of a spatial filter, such as a confocal pinhole, should allow for blocking the out-of-focus signal; however, the decreased collection efficiency deems this approach suboptimal in MPM as previously discussed [14]. Instead, we here propose an approach based on annular beam shaping. As illustrated in Figure 1, the fluorescence, F, generated at a plane, , would be more confined to the focal plane when using an annular laser beam when compared to Gaussian beam illumination. Geometrically, the peak irradiance of the laser light at the sample surface can be lowered by distributing the laser energy in a ring instead of a Gaussian beam profile. This lowers the intensity in any given point of the sample while retaining the photon flux in the focal volume and thus reduces the generation of out-of-focus fluorescence contributing to the undesired background. Previously, annular beam shaping has been investigated for confocal microscopy [15] and multiphoton microscopy [16, 17] to improve resolution. The concept of using annular beams to reduce out-of-focus signal is previously not explored.